Abstract
Human DNA libraries were screened for DNA synthesis activity in vitro using purified DNA polymerase α/primase and a viral DNA helicase (simian virus 40 large tumor antigen). Three clones exhibited a high activity distinguishable from the rest. The DNA synthesis was dependent on negative supertwisting and initiated at a unique region in the human DNA insert. Functional subclone DNA fragments which could be shortened to less than 1 kb are located in the initiation region. Binding with a single-stranded DNA-binding protein and digestion with nuclease P1 demonstrated that these DNAs have a highly single-stranded nature at a certain site in a closed circular plasmid. The minimal functional sequences coincide with the single-stranded region and contain a characteristic dinucleotide repeat sequence. These repeats have an extremely low free energy for DNA strand separation and are denned as DNA-unwinding elements, which are frequently observed at regions flanking replication origins in Escherichia coli and Saccharomyces cerevisiae chromosomes. We suggest that such a repeating sequence would have an important role during initiation of DNA replication and function as a site to recruit replication proteins. DNA replication; DNA helicase; dinucleotide repeats; replication protein A; nuclease P1.
| Original language | English |
|---|---|
| Pages (from-to) | 277-284 |
| Number of pages | 8 |
| Journal | Gene |
| Volume | 148 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Oct 21 1994 |
| Externally published | Yes |
Fingerprint
All Science Journal Classification (ASJC) codes
- Genetics
Cite this
Isolation of human DNA-unwinding elements as sites of DNA polymerase α/primase entry. / Pack, Robert A.; Tsurimoto, Toshiki.
In: Gene, Vol. 148, No. 2, 21.10.1994, p. 277-284.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Isolation of human DNA-unwinding elements as sites of DNA polymerase α/primase entry
AU - Pack, Robert A.
AU - Tsurimoto, Toshiki
PY - 1994/10/21
Y1 - 1994/10/21
N2 - Human DNA libraries were screened for DNA synthesis activity in vitro using purified DNA polymerase α/primase and a viral DNA helicase (simian virus 40 large tumor antigen). Three clones exhibited a high activity distinguishable from the rest. The DNA synthesis was dependent on negative supertwisting and initiated at a unique region in the human DNA insert. Functional subclone DNA fragments which could be shortened to less than 1 kb are located in the initiation region. Binding with a single-stranded DNA-binding protein and digestion with nuclease P1 demonstrated that these DNAs have a highly single-stranded nature at a certain site in a closed circular plasmid. The minimal functional sequences coincide with the single-stranded region and contain a characteristic dinucleotide repeat sequence. These repeats have an extremely low free energy for DNA strand separation and are denned as DNA-unwinding elements, which are frequently observed at regions flanking replication origins in Escherichia coli and Saccharomyces cerevisiae chromosomes. We suggest that such a repeating sequence would have an important role during initiation of DNA replication and function as a site to recruit replication proteins. DNA replication; DNA helicase; dinucleotide repeats; replication protein A; nuclease P1.
AB - Human DNA libraries were screened for DNA synthesis activity in vitro using purified DNA polymerase α/primase and a viral DNA helicase (simian virus 40 large tumor antigen). Three clones exhibited a high activity distinguishable from the rest. The DNA synthesis was dependent on negative supertwisting and initiated at a unique region in the human DNA insert. Functional subclone DNA fragments which could be shortened to less than 1 kb are located in the initiation region. Binding with a single-stranded DNA-binding protein and digestion with nuclease P1 demonstrated that these DNAs have a highly single-stranded nature at a certain site in a closed circular plasmid. The minimal functional sequences coincide with the single-stranded region and contain a characteristic dinucleotide repeat sequence. These repeats have an extremely low free energy for DNA strand separation and are denned as DNA-unwinding elements, which are frequently observed at regions flanking replication origins in Escherichia coli and Saccharomyces cerevisiae chromosomes. We suggest that such a repeating sequence would have an important role during initiation of DNA replication and function as a site to recruit replication proteins. DNA replication; DNA helicase; dinucleotide repeats; replication protein A; nuclease P1.
UR - http://www.scopus.com/inward/record.url?scp=0027959908&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0027959908&partnerID=8YFLogxK
U2 - 10.1016/0378-1119(94)90699-8
DO - 10.1016/0378-1119(94)90699-8
M3 - Article
C2 - 7958955
AN - SCOPUS:0027959908
VL - 148
SP - 277
EP - 284
JO - Gene
JF - Gene
SN - 0378-1119
IS - 2
ER -